Simplified accurate method of detecting bacteriuria



United States Patent 3,547,780 SIMPLIFIED ACCURATE METHOD OF DETECTINGBACTERIURIA Frank A. Finnerty, Jr., 6 Langley Place, McLean, Va. 22101,and Arnold C. Johnson, 6103 Seminole St., College Park, Md. 20741 NoDrawing. Filed Mar. 13, 1968, Ser. No. 712,584 Int. Cl.' C12k 1/10; G01n21/06, 33/16 U.S. Cl. 195-1035 Claims ABSTRACT OF THE DISCLOSURE Themethod of producing a dip stick for detecting bacteriuria comprisingapplying to the stick an aqueous solution of sulfanilic acid and an acidselected from the group consisting of citric and oxalic acids, dryingthe stick, applying a second coating of an anhydrous organic solventsolution of a naphthylamine; and the dip stick produced by this method.

HISTORICAL BACKGROUND This invention relates to the use of a combinationof the Griess nitrite test (which has been relegated to a stickprocedure) and the diphenylamine test in detecting bacteriuria. Thisinvention improves the accuracy of the Griess nitrite test bydifferentiation between a truly negative and a falsely negative test.

In recent years, suggestions were made as to the possibility ofdiagnosing and treating patients -,with genitourinary infection beforeonset of symptoms. Recognition and treatment of asymptomatic bacteriuriain pregnant patients not only reduces the incidence of symptomaticinfection in the mother but also reduces the incidence of prematurityand perinatal mortality. Although many investigators have shown thatgenito-urinary infection can be detected before the onset of symptoms,the importance in the pathogenesis of chronic pyelonephritis renalvascular disease or the relationship to prematurity or perinatalmortality requires documentation for accurate and positive predictions.To date, the only true reliable methods for such documentation consistof sophisticated bacteriologic techniques which are not available in theoffices of most practising physicians. It is therefore of utmostimportance that reliable methods of detecting asymptomatic bacteriuriabe available for performance by the practising physician in his office.

In an attempt to simplify the procedure for identifying bacteriuria,several chemical methods have been developed. The Griess nitrite test,which was developed in 1879, has more recently received the mostattention. It is simple to perform and relatively inexpensive. Its maindisadvantage; however, continues to be the high incidence of falsenegative results. The Griess nitrite test for the detection ofasymptomatic bacteriuria is based on the finding that nearly all of thebacterial species which cause asymptomatic bacteriuria reduce nitrate inthe system to nitrite if given sufiicient time. By definition, bacterialspecies such as E. Coli and A. Aerogenes classified in the familyEnterobacteriacae have this characteristic. It has been found that theGriess test, detects nitrite in concentrations of 0.1 ,ug/ml. A falsenegative test can result from (1) the presence of organisms which do notreduce nitrate (rare), or (2) insuflicient substrate. Insufi'icientsubstrate can result from either (1) an insufficiency of nitrate in thediet or (2) utilization of both nitrate and nitrite by the rapid growthof large numbers of organisms.

Performing the Griess nitrite test on the first morning specimen ofurine in asymptomatic patients who do not have nocturia and who are notundergoing catheter drainage or taking excessive amounts of fluid hasinsured sulficient bladder incubation time and has decreased the in-3,547,780 Patented Dec. 15, 1970 cidence of false negative tests. Sincepatients of a lower income bracket may not have adequate nitrate in thediet, a decrease in dietary nitrate could contribute to the incidence offalse negative tests. Admittedly, increasing the oral intake of nitrateslessens this possibility but the accomplishment changing patients dietsis well beyond the scope of most physicians. Similarly, it has beenfound that the addition of nitrate to the urine was of no practicalvalue since at least two hours of incubation time were needed to producethe amount of nitrite which could be detected. One way to simplify thestandard Griess test would be to provide a nitrite indicator stick as asubstitute for the liquid solutions which would be inexpensive tomanufacture and which would have a relatively long shelf life and highsensitivity.

OBJECTS AND SUMMARY tional chemical to the Griess nitrite test whichreduces the high incidence of false negative Griess tests when used forthe detection of bacteriuria.

Still another object of this invention is to provide a simple test forthe determining of bacteriuria which does not require elaboratelaboratory facilities for its utilization.

A further object of this invention is to provide chemical tests whichwill permit early detection of genito-urinary infections prior to theonset of symptoms and thus reduce the incidence of prematurity andperinatal mortality.

In summary therefore, this invention has as its objects, thesimplification and the increasing of accuracy of a method for detectingbacteriuria utilizing the diphenylamine test with either the standardGriess reagent or an improved nitrite indicator of the type described inthis disclosure.

This invention reduces false negative Griess tests by detecting thepresence of or absence of nitrate in urine found to be Griess testnegative. Establishing the presence or absence of nitrate in urineswhich are Griess test negative permits differentiation between a trulynegative and a falsely negative test. A truly negative test (nitratepositive, colony count negative) will show that there is nitrate in thediet which has not been reduced to nitrite by actively growingorganisms. In the prior art, falsely negative Griess tests would fail toshow the presence of nitrite and would suggest (1) either a lack ofsubstrate,

or (2) that the substrate had been completely utilized by rapidlygrowing organisms. Although diphenylamine reagent has long been used forthe purposes of detecting the presence of nitrates and nitrites it hasnot to our knowledge been used in conjunction with the standard Griesstest. We have found that the diphenylamine test for nitrites is lesssensitive than the Griess test and therefore validates the Griess testresults by combining the diphenylamine test therewith in order todifferentiate false negatives from true negative results to therebydecrease the incidence of false negative tests.

To further simplify the method of testing, the diphenylamine test isused in conjunction with a nitrite indicator stick made in accordancewith the following procedural steps.

METHOD OF PREPARING THE STICK The unprepared stick for use in the testcan be strips of paper, wood, or plastic having absorptive qualities andfilter paper strips have been found to be quite suitable. The dip sticksof filter paper, for ease in handling and in detection as well asstorage, should preferably be approximately three inches in length and aquarter of an inch in width, of an inch in thickness and of a fine mat.It is obvious that considerable latitude can be utilized as far as sizeof the dip stick is concerned. When prepared, the end of the dip stickswill be covered with two relatively thing and porous superimposedcoatings.

In preparation of the sticks, individual sticks can be prepared by themethod herein set out or large sheets of paper, etc., can be preparedwhich after treatment will subsequently cut up into small stick sizesfor ease in handling. To this end, an aqueous solution from about 0.25%to about 2% of sulfanilic acid prepared in approximately 10% citric acidis utilized at room temperature. About /2 inch of the end of strips orsheets of filter paper, etc. is dipped into the solution and dried in arelatively high vacuum at 40 C. until substantially all of the moisturehas been removed. Drying by heating over 40 C. is not advised becauseadditional heat causes deterioration of the chemical composition whichhas been applied to the paper.

A second solution of alpha-naphthylamine is prepared in n-heptane in aconcentration of approximately 0.25% to 2% (Table 1).

TABLE l.-CONCENTRATION OF SULFANILIO ACID IN 10 PERCENT CIIRIO ACIDConcentration of alphanaphthylamine in n-heptane 2% 1% 5% 25% 12% 06%03% Tests conducted with 0.1 gJml. KNO2- The previously treated portionof the strips of filter paper which have been dried are now dipped intothe solution of alpha-naphthylamine and subsequently dried atapproximately 40 C. It has been found that a repeat dipping and dryingof this step in the alpha-naphthylamine solution increases sensitivity.The optimum concentrations of the sulfanilic acid and thealpha-naphthylamine in their solutions is approximately 1%.

It is most important that the moisture (H O) content of the papertreated in the first step be held to a minimum because any moistureremaining in the strip will have an adverse effect When the strip issubsequently treated with the second solution causing discoloration andthus make noticeable color change undetectable. In order to keepmoisture to a minimum, it is suggested that some type of desiccant suchas calcium chloride be utilized in the nheptane solution. N-heptane ispreferably used as a solvent for the alpha-napthylamine because it iscommercially quite dry and does not absorb a sufiicient amount ofmoisture from the air to seriously affect the sulfanilic acid treatedstrips by discoloration once they have been dipped into the n-heptanesolution. Solvents in which the alpha-napthylarnine have limitedsolubility and little Water sorbtivity such as other heptanes, octanes,or hexanes or the like may be used but in general these solvents aremore expensive and not nearly as hygroscopically stable.

Upon drying of the strips which have been treated in both solutions, thestrips, if they have not already been cut, are cut to proper size andplaced in a dark colored bottle such as a brown glass bottle in which asmall amount of desiccant has been placed. The bottles are then coveredfor storage. Shelf life has been found to be excellent and littlediscoloration of the strips has been noted even after six monthsstorage.

Applications of the coatings may be by dipping, spraying, brushing,immersing, etc.

ALTERNATE METHOD Instead of utilizing alpha-naphthylamine, the morestable alpha-naphthylamine hydrochloride may be used by dissolving thesame in absolute ethyl alcohol which has calcium chloride therein. Thecalcium chloride is maintained in the ethyl alcohol in order to maintainthe alcohol as dry as possible since absolute alcohol tends to pick upmoisture from the air With relative ease. Other alcohols can be usedsuch as methyl or propyl. As alpanaphthylamine hydrochloride isdifficultly soluble in the ethyl alcohol, a concentration exceeding onepercent is difficultly obtainable. The optimum concentration of thealpha-naphthylamine hydrochloride in ethyl alcohol is about 0.5% and theeffective range runs from approximately 0.25% to about 1% (Table 2).

TABLE 2.CONCENTRATION OF SULFANILIC ACID IN 10 PERCENT OITRIC ACIDConcentration of alpha-naphthylamine hydrochloride in IIHH++ lllllHllllll Illlll Tests conducted with 0.1 gn/m. KNO

When the dip stick is used for the detection of nitrites in urine, theacidity must be great enough to bring the acidity of the dip stick to apH of about 3.8. In practical application, 10% citric acid or oxalicacid was found to be sufficient for the detection of nitrites in urine.It is felt that from about 5% to 20 of oxalic or citric acid will besufficient to push the pH of the dip stick when testing urine to a pH ofapproximately 3.8. It is to be noted that a coloration change actuallytakes place at a pH of 5.2 but peak color development is reachedapproximately at a pH of about 3.8. A minimum pH therefore for testingwould be more acid than 5.2.

TESTS CONDUCTED TABLE 3.COMPARISON OF CHEMICAL AND BACTERIO- LOGICALDATA IN 624 ASYMPTOMA'IIO PATIENTS Chemical tests, Colony count, cleanvoided urines caught ui'ines Diphenyl- Griess amine Negative PositiveOrganisms isolated 554 0 None. 0 7 6 Escheriachia cali.*

1 Aerobacter aerogenes. 10 16 12 Escherichia coli.*

4 Aerobacter aervge'ncs. 3 Escherichia cali. 11 7 3Aerobucter aerogenes.

1 Proteus vulgarix.

* One formerly laracolobactrum species.

The finding of a temperature above one hundred degrees (Without obviousextra genito-urinary etiology), symptoms of a current genito-urinarytract infection, or the history of taking any antibiotic during the pasttwo weeks excluded the patient from the study. A history of priorgenito-urinary tract infection did not exclude patients from the study.

The first morning voided urine was collected by the patient at home andbrought for testing. The only instruction given to the patient was tocollect the urine in.

a nitrite-nitrate free bottle supplied to the patient. On the same day,a mid-stream sample of urine was collected by a nurse. The patient wascleansed with aqueous green soap and the urine was collected while thepatient was in standing position. Both the patients bottle and the onecollected by the nurse were maintained refrigerated until tested.

The first morning urine sample was tested in accordance with thisinvention utilizing the nitrite indicator test and diphenylamine test.The appearance of a pink color on the stick constituted a positive testfor nitrites. The diphenylamine procedure was performed as a ring test.This test is conducted by placing approximately five-tenths ml. (0.5ml.) urine in a 1 ml. pipette and layering the urine over thediphenylamine reagent with a tube held at a forty-five degree angle andthe tip of the pipette about one-quarter of an inch A) above the surfaceof the reagent. Urine was allowed to flow slowly down the side of thetube and over the diphenylamine reagent. Care was taken not to mix thereagent with the urine. The tube was placed on a rack and read after oneminute against a white background. The appearance of a deep blue colorconstituted a positive test for nitrates and/or nitrites. The appearanceof a deep brown color which masked the blue color suggested that theurine and re agent had been mixed and in these cases a repeat test wasnecessary. No color change or the appearance of a slight violetconstituted a negative test. In order not to overlook a delayed colorchange, the tubes were not dis carded for ten minutes.

The control tests were run with the clean caught urines which werediluted into nutrient broth. From the dilutions, dilution pour plateswere made and incubated at 36 C. and kept overnight and the colonieswhich developed were counted. Urines with a colony count of eightythousand bacteria per ml. or greater were cultured on blood agar etc.The bacteria were identified biochemically. When colony count data wascompared to chemical tests, in no instance was there noted a falsepositive or false negative chemical result. If only the standard Griessnitrate test had been used for screening, approximately 25% of the testswould have been misdiagnosed. Thus this inven tion clearly providesgreater accuracy than heretofore afforded.

The diphenylamine solution may vary from about 0.15% to about 0.01%diphenylamine in concentrated sulfuric acid. The preferred concentrationis 0.05% diphenylamine in sulfuric While the invention has beendescribed, it will be understood that it is capable of furthermodifications and this application is intended to cover any variationsused or adaptations of the invention following in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice in the artto which the invention pertains, and as may be applied to the essentialfeatures hereinbefore set forth and as fall within the scope of theinvention or the limits of the appended claims.

Having thus described our invention, what we claim is:

1. The method of preparing a stick material for detecting bacteriuria inurine comprising:

(a) applying to said stick material an aqueous solution of from about0.25% to about 2% of sulfanilic acid and from about 5% to about of anacid selected from the group consisting of citric and oxalic acids toobtain a first coating on said stick material,

(b) drying said first coated stick material at a temperature notexceeding 40 C. until substantially moisture free,

(c) applying to said first coating an anhydrous organic solvent solutionof from about 0.25% to 2% of an amine selected from the group consistingof alphanaphthylamine and alpha-naphthylamine hydrochloride, saidorganic solvent being selected from the group consisting of methyl,ethyl, propyl alcohols and hexane, heptane and octane to obtain a secondcoating superimposed on said first coating,

(d) and drying said second coated stick material at a temperature notexceeding 40 C.

2. The method of claim 1 and wherein.

(a) said amine is alpha-naphthylamine, said organic solvent is heptaneand said alpha-naphthylamine in said heptane is approximately 1%.

3. The method of claim 1 and wherein:

(a) said amine is alpha naphthylamine hydrochloride, said organicsolvent is ethyl alcohol, and said alpha naphthylamine hydrochloride insaid ethyl alcohol is approximately 0.5%.

4. A method of preparing stick material as in claim 1 and including thestep of:

(a) applying at least one additional coating of said anhydrous organicsolvent solution to said stick, and (b) drying said stick with at leastsaid one additional coating at a temperature not exceeding 40 C.

5. The product produced by the method of claim 1.

6. The method of testing for bacteriuria which comprises:

(a) testing a urine sample with a Griess nitrite test composition, and

(b) subsequently testing said sample with from about 0.15 to about 0.01%diphenylamine in concentrated sulfuric acid solution by layering theurine over said acid solution and checking for a color change at theinterface after approximately one minute.

7. The method of claim 6 and wherein:

(a) said concentration of diphenylamine in concentrated sulfuric acid isabout 0.05

8. The method of claim 6 and wherein:

(a) said Griess nitrite test composition is in a stick form and saidstick includes a first moisture free coating consisting essentially ofsultanilic acid and an acid selected from the group consisting of:oxalic acid and citric acid in a quantity sufficient to acidify urine incontact therewith when testing to a pH of less than 5.2, and including(b) a second moisture free dry coating superimposed on said first driedcoating and consisting essentially of an amine selected from the groupconsisting of: alpha-naphthylamine and 'alpha-naphthylaminehydrochloride, and

(c) said coatings being relatively thin and permeable.

9. The method of claim 8 and wherein:

(a) said amine is alpha-naphthylamine.

10. The method of claim 8 and wherein:

(a) said amine is alpha-naphthylamine hydrochloride.

References Cited UNITED STATES PATENTS 2,990,253 6/1961 Smeby 232533,415,718 12/1968 Forkman 252408 3,415,717 12/1968 Avakian l00 OTHERREFERENCES C.A.(I) 61: 10983a.

C.A.(II) 61: 13622c.

P. B. Hawk et al. Practical Physiological Chem, Thirteenth edition, 822,McGraw-Hill, New York, 1954.

F. Feigel: Spot Tests in Organic Analysis, Fifth edition, 168170,Elsevier Publishing, New York, 1956.

J. Fischl and N. Pinto: Clinica Chimica Acta, 2, 527- 533 (1957).

MORRIS O. WOLK, Primary Examiner S. MARANTZ, Assistant Examiner US. Cl.X.R.

